Immobilization of a recombinant Escherichia coli producing a thermostable alpha-L-rhamnosidase: Creation of a bioreactor for hydrolyses of naringin

An U-L-rhamnosidase (E.C. 3.2.1.40) from a newly discovered thermophilic bacterium was expressed in Escherichia coli BL21 DE3 pRIL cells. The cells were immobilized in Ca2+-alginate beads. The temperature of 50 degrees C used in reactions, appeared to be sufficient for making the mesophilic strain porous enough for the substrate to access the cloned thermostable enzyme. Pretreatment of cells with heat or lysozyme prior to bead formation did not improve the results. The best cell concentration (w/w) for bead preparation was found to be 0.0 192 g ml(-1) and stability of beads increased if CaCl2 concentration in buffers and substrate was kept at 50 mM. In a 60 min assay, the optimal pH of the entrapped cells was found to be 7.8 and the optimal temperature 60 degrees C. By packing the beads in a column, a bioreactor for production Of L-rhamnose from naringin was created. Full degradation of 7.9 mM naringin could be reached by running the reactor at 1 ml min(-1) at 50 degrees C. The optimal running temperature of the reactor was found to be 50 degrees C and the reactor was fully stable over 3 days at that temperature. On the fourth day, substrate degradation capacity had decreased by 10-15%. (c) 2006 Elsevier Inc. All rights reserved

Reduction of <i>Campylobacter </i>on broiler carcasses using slurry ice

Reduction of Campylobacter on broiler meat is crucial for decreasing the number of campylobacteriosis cases. A module (IceGun®) that sprays slurry ice on carcasses has been developed to enhance the chilling capacity in poultry slaughterhouses and may, in addition, help reduce the bacterial load. The present study investigated how slurry ice (IceGun®) affects the Campylobacter load on broiler carcasses. Neck skin pool samples from IceGun® treated (n = 840) and control carcasses (n = 840) were sampled in a poultry slaughterhouse under industrial working conditions from September 2022 to January 2023 and assessed for reduction of Campylobacter quantitatively and qualitatively. Campylobacter counts were significantly lower using the IceGun® by 0.44 log10 CFU/g (CI: 0.31–0.56). The number of Campylobacter-positive samples was significantly lower for the IceGun® treated carcasses than the control carcasses when the cut-off was set at 100 CFU/g, but not at 1000 CFU/g. The present study demonstrated that the IceGun® reduces the Campylobacter contamination level on poultry neck skin, but further modifications of the IceGun® are desirable to achieve a greater reduction of Campylobacter to lower infection risk to the consumer

Two novel cyclodextrin-degrading enzymes isolated from thermophilic bacteria have similar domain structures but differ in oligomeric state and activity profile

In this paper, we present the expression and characterization of two novel enzymes from the a-amylase family exhibiting cyclomaltodextrinase specificity. The nucleotide sequences encoding the enzymes were isolated from the genomic DNA of two thermophilic bacterial strains originating from Icelandic hot springs and belonging to the genera Anoxybacillus (AfCda13) and Laceyella (LsCda13). The genes were amplified using a consensus primer strategy utilizing two of the four conserved regions present in glycoside hydrolase family 13. No identifiable signal peptides were present in open reading frames encoding the enzymes, indicating an intracellular location of both enzymes, and their physiological function to be intracellular cyclodextrin degradation. The domain structures of both enzymes were also similar, including an N-terminal domain, the catalytic module composed of the A- and B-domains, and a C-terminal domain. Despite the similarity in domain composition, the two enzymes displayed differences in the oligomeric state with AfCda13 being a dimeric protein, whereas LsCda13 was monomeric. The two enzymes also displayed significantly different activity profiles, despite being active on the same range of substrates. It was shown that the enzyme displaying the highest activity on cyclodextrin was dimeric (AfCda13). Moreover, a fraction of the dimeric enzyme could be converted to a monomeric state in the presence of KCl and this fraction retained only 23% of its activity on a-cyclodextrin while its activity on starch was not significantly affected, indicating that the oligomeric state is an important factor for a high activity on cyclodextrin substrates

Novel Members of Glycoside Hydrolase Family 13 Derived from Environmental DNA▿

Starch and pullulan-modifying enzymes of the α-amylase family (glycoside hydrolase family 13) have several industrial applications. To date, most of these enzymes have been derived from isolated organisms. To increase the number of members of this enzyme family, in particular of the thermophilic representatives, we have applied a consensus primer-based approach using DNA from enrichments from geothermal habitats. With this approach, we succeeded in isolating three new enzymes: a neopullulanase and two cyclodextrinases. Both cyclodextrinases displayed significant maltogenic amylase side activity, while one showed significant neopullulanase side activity. Specific motifs and domains that correlated with enzymatic activities were identified; e.g., the presence of the N domain was correlated with cyclodextrinase activity. The enzymes exhibited stability under thermophilic conditions and showed features appropriate for biotechnological applications